Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/14—Pretreatment of feeding-stuffs with enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/189—Enzymes

Definitions

• the present invention relates to isolated polypeptides having acetylxylan esterase activity and isolated polynucleotides encoding the polypeptides
• the invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides
• Plant cell wall polysaccharides constitute generally 90% of the plant cell wall and can be divided into three groups cellulose, hemicellulose and pectin Cellulose represents the major constituent of cell wall polysaccharides Hemicelluloses are the second most abundant constituent of plant cell walls The major hemicellulose polymer is xylan The structure of xylans found in cell walls of plants can differ significantly depending on their origin, but they always contain a beta-1 ,4-l ⁇ nked D-xylose backbone The beta-1,4-l ⁇ nked D-xylose backbone can be substituted by various side groups, such as L-a ⁇ binose, D-galactose, acetyl, feruloyl p-coumaroyl, and glucuronic acid residues
• the biodegradation of the xylan backbone depends on two classes of enzymes endoxylanases and beta-xylosidases Endoxylanases (EC 32 1 8) cleave the xylan backbone into smaller oligosaccharides, which can be further degraded to xylose by beta-xylosidases (EC 3 2 1 37)
• Other enzymes involved in the degradation of xylan include, for example, acetylxylan esterase, arabinase, alpha-glucuronidase, feruloyl esterase, and p-coumaric acid esterase
• Acetylxylan esterase (EC 3 1 1 6) removes the O-acetyl groups from positions 2 and/or 3 on the beta-D-xylopyranosyl residues of acetylxylan
• Acetylxylan plays an important role in the hydrolysis of xylan because the acetyl side groups can interfere sterically with the approach of enzymes that cleave the backbone Removal of the acetyl side groups facilitates the action of endoxylanases
• a classification system for carbohydrate esterases based on sequence similarity, has led to the definition of 13 families, seven of which contain acetylxylan esterases (Hennssat B , 1991 , Bochem J 280 309-316, and Henrissat and Bairoch, 1996, Biochem J 316 695-696) Margolles-Clark et al , 1996, Eur J Biochem 237 553-560, disclose an acetylxylan esterase
• the present invention relates to isolated polypeptides having acetylxylan esterase activity selected from the group consisting of
• polypeptide encoded by a polynucleotide that hybridizes under at least medium-high stringency conditions with ( ⁇ ) the mature polypeptide coding sequence of SEQ ID NO 1 , (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1 , or (in) a full-length complementary strand of ( ⁇ ) or ( ⁇ ), (C) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 75% identity to the mature polypeptide coding sequence of SEQ ID NO 1, and
• a va ⁇ ant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO 2
• the present invention also relates to isolated polynucleotides encoding polypeptides having acetylxylan esterase activity, selected from the group consisting of
• the present invention also relates to nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods of producing a polypeptide having acetylxylan esterase activity
• the present invention also relates to methods of inhibiting the expression of a polypeptide having acetylxylan esterase activity in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention
• dsRNA double-stranded inhibitory RNA
• dsRNA double-stranded inhibitory RNA
• the present invention also relates to methods for degrading a xylan-containing material with a polypeptide having acetylxylan esterase activity
• the present invention also relates to plants comprising an isolated polynucleotide encoding a polypeptide having acetylxylan esterase activity
• the present invention also relates to methods of producing a polypeptide having acetylxylan esterase, comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide having acetylxylan esterase activity under conditions conducive for production of the polypeptide, and (b) recovenng the polypeptide
• the present invention further relates to nucleic acid constructs comprising a gene encoding a protein, wherein the gene is operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO 2, wherein the gene is foreign to the nucleotide sequence
• nucleic acid constructs comprising a gene encoding a protein, wherein the gene is operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO 2, wherein the gene is foreign to the nucleotide sequence
• Figures 1A and 1B show the genomic DNA sequence and the deduced amino acid sequence of a Humicola insolens DSM 1800 CE1 acetylxylan esterase (SEQ ID NOs 1 and 2, respectively) Predicted intronic sequences are underlined in bold
• Figure 2 shows a restriction map of pMMar ⁇
• Figure 3 shows a restriction map of pH ⁇ nsAXE2
• Acetylxylan esterase activity is defined herein as a carboxylesterase activity (EC 3 1 1 72) that catalyses the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha- napthyl acetate, and p-nitrophenyl acetate
• acetylxylan esterase activity is determined according to the procedure described herein in the Examples
• One unit of acetylxylan esterase activity is defined as the amount of enzyme capable of releasing 1 ⁇ mole of p-nitrophenolate anion per minute at pH 5, 25°C
• polypeptides of the present invention have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the acetylxylan esterase activity of the mature polypeptide of SEQ ID NO 2
• Family CE1 or CE1 is defined herein as a polypeptide falling into the carbohydrate esterase Family according to Coutinho and
• xylan-containing material is defined herein as any material comprising xylan as a constituent Xylan is a plant cell wall polysacchande containing a backbone of beta-1 ,4-l ⁇ nked xylose residues Side chains of 4-O-methylglucuronic acid and arabinose are generally present in varying amounts, together with acetyl and feruloyl groups Xylan is a major constituent of hemicellulose
• Isolated polypeptide refers to a polypeptide that is isolated from a source In a preferred aspect, the polypeptide is at least 1 % pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE
• substantially pure polypeptide denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0 5% by weight of other polypeptide material with which it is natively or recombinant ⁇ associated It is, therefore, preferred that
• Mature polypeptide The term "mature polypeptide" is defined herein as a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylate, phosphorylation, etc
• the mature polypeptide is amino acids 20 to 377 of SEQ ID NO 2 based on the SignalP program (Nielsen et al , 1997, Protein Engineering 10 1-6) that predicts amino acids 1 to 19 of SEQ ID NO 2 are a signal peptide
• Mature polypeptide coding sequence is defined herein as a nucleotide sequence that encodes a mature polypeptide having acetylxylan esterase activity
• the mature polypeptide coding sequence is nucleotides 58 to 1266 of SEQ ID NO 1 based on the SignalP program (Nielsen ef al , 1997, supra) that predicts nucleotides 1 to 57 of SEQ ID NO 1 encode a signal peptide
• Identity The relatedness between two ammo acid sequences or between two nucleotide sequences is described by the parameter "identity"
• the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algo ⁇ thm (Needleman and Wunsch, 1970, J MoI Biol 48 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS The European Molecular Biology Open Software Suite, Rice et al , 2000, Trends in Genetics 16 276-277), preferably version 30 0 or later
• the optional parameters used are gap open penalty of 10, gap extension penalty of 05, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix
• the output of Needle labeled "longest identity * (obtained using the -nobrief option) is used as the percent identity and is calculated as follows (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
• the degree of identity between two deoxy ⁇ bonucleotide sequences is determined using the Needleman-Wunsch algorithm
• EMBOSS package (EMBOSS The European Molecular Biology Open Software Suite, Rice et al 2000, supra), preferably version 3 0 0 or later
• the optional parameters used are gap open penalty of 10, gap extension penalty of 05, and the EDNAFULL (EMBOSS version of NCBI NUC44) substitution matrix
• the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of
• homologous sequence is defined herein as a predicted protein that has an E value (or expectancy score) of less than 0 001 in a tfasty search (Pearson, W R , 1999, in Bomformatics Methods and Proto ⁇ ls, S Misener and S A Krawetz, ed , pp 185-219) with the Humicola insolens acetylxylan esterase of SEQ ID NO 2 or the mature polypeptide thereof
• Polypeptide fragment is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of SEQ ID NO 2, or a homologous sequence thereof, wherein the fragment has acetylxylan esterase activity
• a fragment contains at least 310 amino acid residues, more preferably at least 325 ammo acid residues, and most preferably at least 340 amino acid residues, of the mature polypeptide of SEQ ID NO 2 or a homologous sequence thereof
• Subsequence is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5' and/or 3' end of the mature polypeptide coding sequence of SEQ ID NO 1 , or a homologous sequence thereof, wherein the subsequence encodes a polypeptide fragment having acetylxylan esterase activity
• a subsequence contains at least 930 nucleotides, more preferably at least 975 nucleotides, and most preferably at least 1020 nucleotides of the mature polypeptide coding sequence of SEQ ID NO 1 or a homologous sequence thereof
• allelic variant denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus Allelic va ⁇ ation arises naturally through mutation, and may result in polymorphism within populations Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered ammo acid sequences
• allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene
• Isolated polynucleotide refers to a polynucleotide that is isolated from a source
• the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis
• substantially pure polynucleotide refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems
• a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 05% by weight of other polynucleotide material with which it is natively or recombinant ⁇ associated
• a substantially pure polynucleotide may, however, include naturally occur ⁇ ng 5' and 3' untranslated regions, such as promoters and terminators It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at
• Coding sequence means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product
• the boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA
• the coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence cDNA:
• cDNA is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell cDNA lacks intron sequences that may be present in the corresponding genomic DNA
• the initial, primary RNA transcript is a precursor to mRNA that is processed through a senes of steps before appearing as mature spliced mRNA These steps include the removal of intron sequences by a process called splicing cDNA derived
• nucleic acid construct refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic
• nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention
• control sequences The term "control sequences * is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention
• Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other
• Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator
• the control sequences include a promoter, and transc ⁇ ptional and translational stop signals
• the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide
• operably linked denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide
• expression includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transc ⁇ ptional modification, translation, post-translational modification, and secretion Expression vector
• expression vector is defined herein as a linear or circular DNA molecule that compnses a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression
• host cell includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention
• Modification means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO 2, or a homologous sequence thereof, as well as genetic manipulation of the DNA encoding such a polypeptide
• the modification can be a substitution, a deletion, and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains
• artificial variant means a polypeptide having acetylxylan esterase activity produced by an organism expressing a modified polynucleotide sequence of the mature polypeptide coding sequence of SEQ ID NO 1 , or a homologous sequence thereof
• the modified nucleotide sequence is obtained through human intervention by modification of the polynucleotide sequence disclosed in SEQ ID NO 1 , or a homologous sequence thereof Detailed Description of the Invention
• the present invention relates to isolated polypeptides comprising an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO 2 of preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99%, which have acetylxylan esterase activity (hereinafter "homologous polypeptides”)
• the homologous polypeptides have an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO 2
• a polypeptide of the present invention preferably compnses the amino acid sequence of SEQ ID NO 2 or an allelic variant thereof, or a fragment thereof having acetylxylan esterase activity
• the polypeptide comprises the amino acid sequence of SEQ ID NO 2
• the polypeptide comprises the mature polypeptide of SEQ ID NO 2
• the polypeptide compnses amino acids 20 to 377 of SEQ ID NO 2, or an allelic variant thereof, or a fragment thereof having acetylxylan esterase activity
• the polypeptide compnses amino acids 20 to 377 of SEQ ID NO 2
• the polypeptide consists of the ammo acid sequence of SEQ ID NO 2 or an allelic variant thereof, or a fragment thereof having acetylxylan esterase activity
• the polypeptide consists of the amino acid sequence of SEQ ID NO 2
• the polypeptide consists of the mature polypeptide of SEQ ID NO 2 In
• a genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having acetylxylan esterase activity
• Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques
• DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier mate ⁇ al
• the carrier material is preferably used in a Southern blot
• hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO 1 , the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1, its full-length complementary strand, or a subsequence thereof, under very low to very high stringency conditions Molecules to which the nucleic acid probe hybndizes under these conditions can be detected using, for example, X-ray film
• the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO 1
• the nucleic add probe Is nucleotides 58 to 1266 of SEQ ID NO 1
• the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO 2, or a subsequence thereof
• the nucleic acid probe is SEQ ID NO 1 In another preferred
• very low to very high stringency conditions are defined as prehyb ⁇ dization and hybridization at 42 0 C in 5X SSPE, 0 3% SDS, 200 ⁇ g/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally
• the carrier material is finally washed three times each for 15 minutes using 2X SSC, 0 2% SDS preferably at 45°C (very low st ⁇ ngency), more preferably at 50°C (low stringency), more preferably at 55°C (medium stringency), more preferably at 60 0 C (medium-high stringency), even more preferably at 65 0 C (high stringency), and most preferably at 70 0 C (very high stringency)
• stringency conditions are defined as prehybridization, hybridization, and washing post- hybndization at about 5°C to about 10 0 C below the calculated T m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48 1390) in 0 9 M NaCI, 0 09 M Tris-HCI pH 7 6, 6 mM EDTA, 0 5% NP- 40, 1X Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0 1 mM ATP, and 02 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally
• the carrier mate ⁇ al is washed once in ⁇ X SCC plus 0 1% SDS for 15 minutes and twice each for 15 minutes using 6X SSC at
• the present invention relates to isolated polypeptides having acetylxylan esterase activity encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO 1 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99%, which encode a polypeptide having acetylxylan esterase activity See polynucleotide section herein
• the present invention relates to artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO 2, or a homologous sequence thereof
• amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein, small deletions, typically of one to about 30 ammo acids, small amino- or carboxyl- terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain
• conservative substitutions are within the group of basic amino acids (argimne, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagme), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, se ⁇ ne, threonine and methionine)
• Amino acid substitutions that do not generally alter specific activity are known in the art and are descnbed, for example, by H Neurath and R L Hill, 1979, In, The Proteins, Academic Press, New York
• the most commonly occurring exchanges are Ala/Ser, Val/lle, Asp/Glu Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/A
• non-standard amino acids such as 4-hydroxyprol ⁇ ne, 6- ⁇ /-methyl lysine, 2-am ⁇ no ⁇ sobutyr ⁇ c acid, isovaline, and alpha-methyl serine
• a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues "Unnatural amino acids * have been modified after protein synthesis, and/or have a chemical structure in their side cha ⁇ n(s) different from that of the standard amino acids
• Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroprohne, 3- and 4-methyl ⁇ roline, and 3,3-dimethyl ⁇ roline
• ammo acid changes are of such a nature that the physico- chemical properties of the polypeptides are altered
• amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, 5 change the pH optimum, and the like
• Essential ammo acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244 1081-1085) In the latter technique, single alanine mutations are introduced at every residue in the molecule, and
• the resultant mutant molecules are tested for biological activity (i e , acetylxylan esterase activity) to identify amino acid residues that are critical to the activity of the molecule See also, Hilton et a/ , 1996, J Biol Chem 271 4699-4708
• biological activity i e , acetylxylan esterase activity
• the active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic
• Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 53-57, Bowie and Sauer, 1989, Proc
• Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagemzed polypeptides expressed by host cells (Ness et a/ , 1999, Nature Biotechnology 17 893-896) Mutagemzed DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art These
• 35 methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure
• the total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO 2, such as amino acids 20 to 377 of SEQ ID NO 2, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1
• a polypeptide of the present invention may be obtained from microorganisms of any genus
• the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted
• the polypeptide obtained from a given source is secreted extracellularly
• a polypeptide having acetylxylan esterase activity of the present invention may be a bacterial polypeptide
• the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Ent ⁇ rococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having acetylxylan esterase activity, or a Gram negative bacterial polypeptide such as an E coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacte ⁇ um, Fusobacterium, llyobacter, Neisseria, or Ureaplasma polypeptide having acetylxylan esterase activity
• the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brews, Bacillus ⁇ rculans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lichewformis, Bacillus megatenum, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thurmgiensis polypeptide having acetylxylan esterase activity
• the polypeptide is a Streptococcus eqwsimilis, Streptococcus pyogenes, Streptococcus ubens, or Streptococcus equi subsp Zooepidemicus polypeptide having acetylxylan esterase activity
• the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces gnseus, or Streptomyces lividans polypeptide having acetylxylan esterase activity
• a polypeptide having acetylxylan esterase activity of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having acetylxylan esterase activity, or more preferably a filamentous fungal polypeptide such as an Acremonium, Agancus, Alternana, Aspergillus, Aureobasidium, Botryospaena, Cenponopsis, Chaetomidium, Chrysospo ⁇ um, Clavicep
• the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyven, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having acetylxylan esterase activity
• the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamon, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosponum keratinophilum, Chrysosponum lucknowense, Chrysospo ⁇ um tropicum, Chrysosponum merdanum, Chrysosponum mops, Chrysosponum pannicola, Chrysosponum queenslandicum, Chrysosponum zonatum, Fusanum bactndioides, Fusartum cerealis, Fusanum crookwellense, Fusanum culmorum, Fusanum graminearum, Fusanum grammum, Fusanum heterosporum, Fu
• the polypeptide is a Humicola insolens polypeptide having acetylxylan esterase activity
• the polypeptide is a Humicola insolens DSM 1800 polypeptide having acetylxylan esterase activity, e g , the polypeptide comprising the mature polypeptide of SEQ ID NO 2
• CBS Schimmelcultures
• NRRL Northern Regional Research Center
• polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e g , soil, composts, water, etc ) using the above-mentioned probes Techniques for isolating microorganisms from natural habitats are well known in the art
• the polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism
• the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e g , Sambrook et al , 1 ⁇ 89, supra)
• Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof
• a fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention
• Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same ⁇ romoter(s) and terminator
• a fusion polypeptide can further comprise a cleavage site Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide having acetylxylan esterase activity from the fusion protein
• cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al , 2003, J lnd Microbiol Btotechnol 3 568-76, Svetina et al , 2000, J Biotechnol 76 245-251 , Rasmussen- Wilson et al , 1997, Appl Environ Microbiol 63 3488-3493, Ward et al , 1995, Biotechnology 13 498-503, and Contreras et al , 1991 , Biotechnology 9 378-381), an Me-(GIu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine
• the present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides having acetylxylan esterase activity of the present invention
• nucleotide sequence comp ⁇ ses or consists of SEQ ID NO 1 the nucleotide sequence comprises or consists of the sequence contained in plasmid pH ⁇ nsAXE2 which is contained in E coli NRRL B-50076
• nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO 1
• nucleotide sequence comprises or consists of the mature polypeptide coding sequence contained in plasmid pH ⁇ nsAXE2 which is contained in E coli NRRL B-50076
• the present invention also encompasses nucleotide sequences that encode polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO 2 or the mature polypeptide thereof, which differ from SEQ ID NO 1 or the mature polypeptide coding sequence thereof
• the present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NO 1 , in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO 2
• the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof
• the cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e g , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features See, e g , lnnis et al , 1990, PCR A Guide to Methods and Application, Academic Press, New York
• Other nucleic acid amplification procedures such as hgase chain reaction (LCR), hgated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used
• LCR hgase chain reaction
• LAT hgated activated transcription
• NASBA nucleotide sequence-based amplification
• nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide
• substantially similar to the polypeptide refers to non-naturally occurring forms of the polypeptide
• the va ⁇ ant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NO 1, e g , a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another ammo acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give ⁇ se to a different amino acid sequence
• the present invention also relates to isolated polynucleotides encoding polypeptides of the present invention, which hybridize under very low stnngency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO 1, ( ⁇ ) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1 , or ( ⁇ ) a full- length complementary strand of (i) or ( ⁇ ), or allelic variants and subsequences thereof (Sambrook et al , 1989, supra), as defined herein
• the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO 1
• the present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under very low, low, medium, medium-high, high, or very
• the present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences
• An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector
• the techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art
• the control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention
• the promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide
• the promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell
• suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell are the promoters obtained from the E coli lac operon, Streptomyces coelicolor agarase gene (dag A), Bacillus subtihs levansucrase gene (sacS), Bacillus licheniformis alpha-amylase gene (a/nyt
• promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusa ⁇ um venenatum amyloglucosidase (WO 00/56900), Fusa ⁇ um venenatum Dana (WO 00/56900), Fusarium
• Saccharomyces cerevisiae enolase ENO-1
• Saccharomyces cerevisiae galactokinase GAL1
• Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3- phosphate dehydrogenase ADH1 , ADH2/GAP
• Saccharomyces cerevisiae triose phosphate isomerase TPI
• Saccharomyces cerevisiae metallothionein CUP1
• Saccharomyces cerevisiae 3-phosphoglycerate kinase Other useful promoters for yeast host cells are described by Romanos et a/ , 1992, Yeast 8 423-488
• the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription
• the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide Any terminator that is functional in the host cell of choice may be used in the present invention
• Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease
• Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase Other useful terminators for yeast host cells are descnbed by Romanos et at , 1992, supra
• the control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell
• the leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide Any leader sequence that is functional in the host cell of choice may be used in the present invention
• Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus niduians triose phosphate isomerase
• Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP)
• ENO-1 Saccharomyces cerevisiae enolase
• Saccharomyces cerevisiae 3- phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
• Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase ADH2/GAP
• control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention
• Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus niduians anthranilate synthase, Fusarium oxysporum trypsin- hke protease, and Aspergillus niger alpha-glucosidase
• the control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway
• the 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence
• the foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide
• any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, / e , secreted Into a culture medium
• Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin,
• Bacillus licheniformis beta-lactamase Bacillus stearothermophilus neutral proteases
• TAKA amylase Aspergillus niger neutral amylase
• Aspergillus niger glucoamylase Aspergillus niger glucoamylase
• Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Sacchammyces cerevisiae invertase
• the signal peptide comprises or consists of ammo acids 1 to 19 of SEQ ID NO 2
• the signal peptide coding sequence comprises or consists of nucleotides 1 to 57 of SEQ ID NO 1
• the control sequence may also be a propeptide coding sequence that codes for an amino acid sequence positioned at the amino terminus of a polypeptide
• the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases)
• a propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide
• the propeptide coding sequence may be obtained from the genes for
• Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT),
• Saccharomyces cerevisiae alpha-factor Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and
• Myceliophthora thermophila laccase (WO 95/33836) Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence
• regulatory systems include the lac, tac, and trp operator systems In yeast, the ADH2 system or GAL 1 system may be used In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences
• regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals In these cases, the nucleotide sequence encoding the
• the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals
• vanous nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites
• a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comp ⁇ sing the sequence into an approp ⁇ ate vector for expression In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression
• the recombinant expression vector may be any vector (e g , a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bnng about expression of the nucleotide sequence
• the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced
• the vectors may be linear or closed circular plasmids
• the vector may be an autonomously replicating vector, / e , a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e g , a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome
• the vector may contain any means for assunng self-replication Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated
• a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used
• the vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells
• a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to
• bacterial selectable markers are the dal genes from Bacillus s ⁇ btilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance
• Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3
• Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothncin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orot ⁇ d ⁇ ne-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anth
• the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination
• the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise locat ⁇ on(s) in the chromosome(s)
• the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination
• the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell
• the integrational elements may be non-encoding or encoding nucleotide sequences
• the vector may be integrated into
• the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question
• the ongin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell
• the term "origin of replication" or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo
• Examples of bacterial origins of replication are the ongins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E coll, and pUBHO pEi94, pTAiO ⁇ O, and pAM ⁇ i permitting replication in Bacillus
• Examples of ongins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6
• Examples of ongins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems ef a/ , 1991, Gene 98 61-67, Cullen ef a/ , 1987, Nucleic Acids
• Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883 More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product
• An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent
• the procedures used to hgate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e g , Sambrook et al , 1989, supra)
• the present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides
• a vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier
• the term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source
• the host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e g , a prokaryote or a eukaryote
• the prokaryotic host cell may be any Gram positive bactenum or a Gram negative bactenum Gram positive bacteria include, but not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus,
• Lactococcus, Clostridium, Geoba ⁇ llus, and Oceanoba ⁇ llus Gram negative bacteria include, but not limited to, £ coli, Pseudomonas, Salmonella, Campylobacter,
• the bacterial host cell may be any Bacillus cell Bacillus cells useful in the practice of the present invention include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefa ⁇ ens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus lichemformis, Bacillus megatenum, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thu ⁇ ngiensis cells
• the bactenal host cell is a Bacillus amyloliquefa ⁇ ens,
• Bacillus lentus, Bacillus lichemformis, Bacillus stearothermophilus or Bacillus subtilis cell In a more preferred aspect, the bacterial host cell is a Bacillus amyloliquefa ⁇ ens cell In another more preferred aspect, the bacterial host cell is a Bacillus clausii cell In another more preferred aspect, the bacterial host cell is a Bacillus lichemformis cell In another more preferred aspect, the bactenal host cell is a Bacillus subtilis cell
• the bacterial host cell may also be any Streptococcus cell Streptococcus cells useful in the practice of the present invention include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp Zooepidemicus cells
• the bactenal host cell is a Streptococcus equisimilis cell
• the bacterial host cell is a Streptococcus pyogenes cell
• the bacterial host cell is a Streptococcus uberis cell
• the bacterial host cell is a Streptococcus equi subsp Zooepidemicus cell
• the bactenal host cell may also be any Streptomyces cell Streptomyces cells useful in the practice of the present invention include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitihs, Streptomyces coehcolor, Streptomyces griseus, and Streptomyces hvidans cells
• the bacterial host cell is a Streptomyces achromogenes cell In another preferred aspect, the bacterial host cell is a Streptomyces avermitihs cell In another preferred aspect, the bacterial host cell is a Streptomyces coehcolor cell In another preferred aspect, the bacterial host cell is a Streptomyces griseus cell In another preferred aspect, the bacterial host cell is a Streptomyces lividans cell
• the introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e g , Chang and Cohen, 1979, Molecular General Genetics 168 111-115), by using competent cells (see, e g , Young and Spizizen, 1961 , Journal of Bacteriology 81 823-829, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56 209-221), by electroporation (see, e g , Shigekawa and Dower, 1988, Biotechniques 6 742-751), or by conjugation (see, e g , Koehler and Thome, 1987, Journal of Bacteriology 169 5271-5278)
• the introduction of DNA into an E coll cell may, for instance, be effected by protoplast transformation (see, e g , Hanahan, 1983, J MoI Biol 166 557-580) or electroporation (see, e g , Dower ef a
• the host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell
• the host cell is a fungal cell "Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chyt ⁇ diomycota, and Zygomycota (as defined by Hawksworth et al , In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al , 1995, supra, page 171) and all mitospo ⁇ c fungi (Hawksworth etal , 1995, supra)
• the fungal host cell is a yeast cell "Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes) Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as
• the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell
• the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasn, Saccharomyces kluyven, Saccharomyces norbensis, or Saccharomyces oviformis cell
• the yeast host cell is a Kluyveromyces lactis cell
• the yeast host cell is a Yarrowia lipolytica cell
• the fungal host cell is a filamentous fungal cell "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al , 1995, supra)
• the filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides
• Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic
• vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative
• the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Cenponopsis, Chrysosponum, Coprinus, Conolus, Cryptococcus, Filibasidium, Fusanum, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Pae ⁇ lomyces, Peni ⁇ llium, Phanerochaete, Phlebia, Piromyces, Pleurot ⁇ s, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell
• the filamentous fungal host cell is an Aspergillus awamon, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell
• the filamentous fungal host cell Is a Fusarium bactridloides, F ⁇ sarium cerealis, Fusarium crookweltense, Fusanum culmorum, Fusanum graminearum, Fusanum graminum, Fusanum heterosporum, Fusanum negundi, Fusanum oxysporum, Fusanum reticulatum, Fusanum roseum, Fusanum sambu ⁇ num, Fusanum sarcochroum, Fusanum sporot ⁇ chioides, Fusarium sulphure ⁇ m, Fusanum torul
• Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se Suitable procedures for transformation of Aspergillus and Tnchoderma host cells are desc ⁇ bed in EP 238 023 and Yelton et at , 1984, Proceedings of the National Academy of Sciences USA 81 1470-1474 Suitable methods for transforming Fusanum species are described by Malardier ef a/ , 1989, Gene 78 147-156, and WO 96/00787 Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J N and Simon, M I , editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, lnc , New York, Ho et al , 1983, Journal of Bacteriology 153 163, and Hinnen ef al , 1978, Proceedings of the National Academy of Sciences USA 75 1920 Methods of Production
• the present invention also relates to methods of producing a polypeptide of the present invention, compnsing (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide, and (b) recove ⁇ ng the polypeptide
• the cell is of the genus Humicola
• the cell is Humicola insolens
• the cell is Humicola insolens DSM 1800
• the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell, as descnbed herein, under conditions conducive for production of the polypeptide, and (b) recovenng the polypeptide
• the present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO 1 , wherein the mutant nucleotide sequence encodes a polypeptide that comprises or consists of the mature polypeptide of SEQ ID NO 2, and (b) recovering the polypeptide
• the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art
• the cell may be cultivated by shake flask cultivation, and small- scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industnal fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated
• the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art Suitable media are available from commercial suppliers or may be prepared according to published compositions (e g , in catalogues of the Amencan Type Culture Collection) If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium If the polypeptide is not secreted into the medium, it can be recovered from cell lysates
• polypeptides may be detected using methods known in the art that are specific for the polypeptides These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate For example, an enzyme assay may be used to determine the activity of the polypeptide as descnbed herein
• the resulting polypeptide may be recovered using methods known in the art
• the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation
• the polypeptides of the present invention may be purified by a vanety of procedures known in the art including, but not limited to, chromatography (e g , ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e g , preparative isoelectric focusing), differential solubility (e g , ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e g , Protein Purification, J -C Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides
• the present invention also relates to plants, e g , a transgenic plant, plant part, or plant cell, comprising an isolated polynucleotide encoding a polypeptide having acetylxylan esterase activity of the present invention so as to express and produce the polypeptide in recoverable quantities
• the polypeptide may be recovered from the plant or plant part Alternatively, the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e g , improving nutritional value, palatability, and rheological properties, or to destroy an anIFF ⁇ tive factor
• the transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot)
• monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e g , wheat, oats, rye, barley, rice, sorghum, and maize (corn)
• dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana
• plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues compnsing these parts, e g , epidermis, mesophyll, parenchyme, vascular tissues, menstems
• Specific plant cell compartments such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part
• any plant cell whatever the tissue origin, is considered to be a plant part
• plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e g , embryos, endosperms, aleurone and seeds coats
• transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art
• the plant or plant cell Is constructed by incorporating one or more (several) expression constructs encoding a polypeptide of the present invention into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell
• the expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a polypeptide of the present invention operably linked with approp ⁇ ate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice
• the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct
• regulatory sequences such as promoter and terminator sequences and optionally signal or transit sequences is determined, for example, on the basis of
• the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves Regulatory sequences are, for example, described by Tague ef al , 1988, Plant
• the 35S-CaMV, the maize ubiquitin 1 , and the rice actin 1 promoter may be used (Franck et al , 1980, Cell 21 285-294, Christensen ef al , 1992, Plant Mo BoI 18 675-689, Zhang ef a/ , 1991 , Plant Cell 3 1155-1165) organ- specific promoters may be, for example, a promoter from storage sink tissues such as
• the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato
• the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e g , ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals
• a promoter enhancer element may also be used to achieve higher expression of a polypeptide of the present invention in the plant
• the promoter enhancer element may be an intron that is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention
• the selectable marker gene and any other parts of the expression construct may be chosen from those available in the art
• the nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-medtaied transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al , 1990, Science 244 1293, Potrykus, 1990, Biotechnology Z 535, Shimamoto er a/ , 1989, Nature 338 274)
• Agrobactenum tumefa ⁇ ens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants
• the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2 275- 281 , Shimamoto, 1994, Current Opinion Biotechnology 5 158-162, Vasil et al , 1992, Bio/Technology 10 667-674)
• An alternative method for transformation of monocots is based on protoplast transformation as descnbed by Omirulleh ef a/ , 1993, Plant Molecular Biology 21 415-428
• the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well- known in the art Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase
• the present invention also relates to methods of producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide having acetylxylan esterase activity of the present invention under conditions conducive for production of the polypeptide, and (b) recove ⁇ ng the polypeptide Removal or Reduction of Acetylxylan Esterase Activity
• the present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting or deleting a polynucleotide sequence, or a portion thereof, encoding a polypeptide of the present invention, which results in the mutant cell producing less of the polypeptide than the parent cell when cultivated under the same conditions
• the mutant cell may be constructed by reducing or eliminating expression of a nucleotide sequence encoding a polypeptide of the present invention using methods well known in the art, for example, insertions, disruptions, replacements, or deletions
• the nucleotide sequence is inactivated
• the nucleotide sequence to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for the expression of the coding region
• An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, / e , a part that is sufficient for affecting expression of the nucleotide sequence
• Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator
• Modification or inactivation of the nucleotide sequence may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the nucleotide sequence has been reduced or eliminated
• the mutagenesis which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis
• the mutagenesis may be performed by use of any combination of these mutagenizing agents
• Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamme, N-methyl-N'-n ⁇ tro-N- nitrosoguanidine (MNNG), O-methyl hydroxylamme, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues
• UV ultraviolet
• Modification or inactivation of the nucleotide sequence may be accomplished by introduction, substitution, or removal of one or more (several) nucleotides in the gene or a regulatory element required for the transcription or translation thereof
• nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame
• modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art
• the modification may be performed in vivo, i e , directly on the cell expressing the nucleotide sequence to be modified, it is preferred that the modification be performed in vitro as exemplified below
• nucleotide sequence corresponding to the endogenous nucleotide sequence is mutagemzed in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene
• the defective nucleic acid sequence replaces the endogenous nucleotide sequence
• the defective nucleotide sequence also encodes a marker that may be used for selection of transformants in which the nucleotide sequence has been modified or destroyed
• the nucleotide sequence is disrupted with a selectable marker such as those described herein
• modification or inactivation of the nucleotide sequence may be performed by established anti-sense or RNAi techniques using a sequence complementary to the nucleotide sequence More specifically, expression of the nucleotide
• the present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a nucleotide sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell
• the polypeptide-deficient mutant cells so created are particularly useful as host cells for the expression of native and/or heterologous polypeptides Therefore, the present invention further relates to methods of producing a native or heterologous polypeptide comprising (a) cultivating the mutant cell under conditions conducive for production of the polypeptide, and (b) recovering the polypeptide
• heterologous polypeptides is defined herein as polypeptides that are not native to the host cell, a native protein in which modifications have been made to alter the native sequence, or a native protein whose expression is quantitatively altered as a result of a manipulation of the host cell by recombinant DNA techniques
• the present invention relates to a method of producing a protein product essentially free of acetylxylan esterase activity by fermentation of a cell that produces both a polypeptide of the present invention as well as the protein product of interest by adding an effective amount of an agent capable of inhibiting acetylxylan esterase activity to the fermentation broth before, during, or after the fermentation has been completed, recovering the product of interest from the fermentation broth, and optionally subjecting the recovered product to further purification
• the present invention relates to a method of producing a protein product essentially free of acetylxylan esterase activity by cultivating the cell under conditions permitting the expression of the product, subjecting the resultant culture broth to a combined pH and temperature treatment so as to reduce the acetylxylan esterase activity substantially, and recovering the product from the culture broth
• the combined pH and temperature treatment may be performed on an enzyme preparation recovered from the culture broth
• the combined pH and temperature treatment may optionally be used in combination with a treatment with an acetylxylan esterase inhibitor
• the methods of the present invention for producing an essentially acetylxylan esterase-free product is of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes
• the enzyme may be selected from, e g , an amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme, oxidoreductase, or plant cell-wall degrading enzyme
• examples of such enzymes include an aminopeptidase, amylase, amyloglucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextnn glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glu
• eukaryotic polypeptides includes not only native polypeptides, but also those polypeptides, e g , enzymes, which have been modified by amino acid substitutions, deletions or additions, or other such modifications to enhance activity, thermostability, pH tolerance and the like
• the present invention relates to a protein product essentially free from acetylxylan esterase activity that is produced by a method of the present invention
• the present invention also relates to methods of inhibiting the expression of a polypeptide of the present invention in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention
• dsRNA double-stranded RNA
• the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length
• the dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA)
• the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription
• the dsRNA is micro RNA (miRNAs) for inhibiting translation
• the present invention also relates to such double-stranded RNA (dsRNA) molecules, compnsing a portion of the mature polypeptide coding sequence of SEQ ID NO 1 for inhibiting expression of a polypeptide in a cell
• dsRNA double-stranded RNA
• the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs
• ssRNA single-stranded RNA
• mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi)
• the dsRNAs of the present invention can be used in gene-silencing therapeutics
• the invention provides methods to selectively degrade RNA using the dsRNAis of the present invention
• the process may be practiced in vitro, ex vivo or in vivo
• the dsRNA molecules can be used to generate a loss-of- function mutation in a cell, an organ or an animal
• Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U S Patent No 6,506,559, U S Patent No 6,511,824, U S Patent No 6,515,109, and U S Patent No 6,489,127
• the present invention also relates to compositions comprising a polypeptide of the present invention
• the compositions are enriched in such a polypeptide
• the term "enriched" indicates that the acetylxylan esterase activity of the composition has been increased, e g , with an enrichment factor of at least 1 1
• the composition may comprise a polypeptide of the present invention as the major enzymatic component, e g , a mono-component composition
• the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodext ⁇ n glycosyltransferase, deoxynbonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,
• polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition
• the polypeptide composition may be in the form of a granulate or a microgranulate
• the polypeptide to be included in the composition may be stabilized in accordance with methods known in the art
• the present invention is also directed to methods for using the polypeptides having acetylxylan esterase activity
• polypeptides of the present invention can be used for degradation or modification of plant cell walls or any xylan-containing material originating from plant cells walls Examples of vanous uses are described below (see, WO 2002/18561 , for other uses)
• the dosage of the polypeptides of the present invention and other conditions under which the preparation is used may be determined on the basis of methods known in the art
• the polypeptides of the present invention are preferably used in conjunction with other xylan degrading enzymes such as xylanases, acetylxylan esterases, arabi ⁇ ofuranosidases, xylosidases, feruloyl esterases, glucuronidases, and a combination thereof, in processes wherein xylan is to be degraded
• xylanases acetylxylan esterases, arabinose groups by alpha- arabinosidases, feruloyl groups by feruloyl esterases, and glucuronic acid groups by alpha-glucuronidases
• oligomers released by the xylanases, or by a combination of xylanases and side branch-hydrolyzing enzymes can be further degraded to free xylose by
• the plant matenal may be degraded in order to improve different kinds of processing, facilitate purification or extraction of components other than the xylans, like purification of beta-glucan or beta-glucan oligomers from cereals, improve the feed value, decrease the water binding capacity, improve the degradability in waste water plants, improve the conversion of, for example, grass and corn to ensilage, etc
• the polypeptides of the present invention may be used in the enzymatic hydrolysis of vanous plant cell wall- derived materials or waste materials, e g , from paper production, or agricultural residues such as wheat-straw, com cobs, corn fiber, whole corn plants, nut shells, grass, vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the like
• the polypeptides may also be used for modifying the viscosity of plant cell wall de ⁇ ved matenal For instance, the polypeptides may be used to reduce the viscosity of xylan-containing matenal,
• the polypeptides of the present invention may also be used with limited activity of other xylanolytic enzymes to degrade xylans for production of oligosaccharides
• the oligosaccharides may be used as bulking agents, like arabinoxylan oligosaccharides released from cereal cell wall material, or of more or less purified arabmoxylans from cereals
• polypeptides of the present invention may also be used in combination with other xylanolytic enzymes to degrade xylans to xylose and other monosaccharides (U S Patent No 5,658,765)
• xylanolytic enzymes to degrade xylans to xylose and other monosaccharides (U S Patent No 5,658,765)
• the released xylose may be converted to other compounds
• polypeptides of the present invention may also be used in lignocellulosic biomass degradation or conversion to fermentable sugars for the production of, for example, fuel, potable ethanol, and/or fermentation products (e g , acids, alcohols, ketones, gases, and the like)
• the polypeptides are preferably used in combination with other xylan degrading enzymes and a cellulase composition (endoglucanase(s), cellob ⁇ ohydrolase(s), and beta-glucos ⁇ dase(s))
• polypeptides of the present invention may be used together with other enzymes like glucanases to improve the extraction of oil from oil- ⁇ ch plant material, like corn-oil from corn-embryos
• the polypeptides of the present invention may also be used in baking to improve the development, elasticity, and/or stability of dough and/or the volume, crumb structure, and/or anti-staling properties of the baked product
• the polypeptides may be used for the preparation of dough or baked products prepared from any type of flour or meal (e g , based on wheat, rye, barley, oat, or maize)
• the baked products produced with a polypeptide of the present invention include bread, rolls, baquettes and the like
• a polypeptide of the present invention may be used as the only or major enzymatic activity, or may be used in combination with other enzymes such as a xylanase, a lipase, an amylase, an oxidase (e g , glucose oxidase, peroxidase), a laccase and/or a protease
• the polypeptides of the present invention may also be used for modification of animal feed and may exert their effect either in vitro (by modifying components of the feed) or in vivo
• the polypeptides may be added to animal feed compositions containing high amounts of arabinoxylans and glucuronoxylans, e g , feed containing cereals such as barley, wheat, rye, oats, or maize
• When added to feed the polypeptide will improve the in vn/o break-down of plant cell wall material partly due to a reduction of intestinal viscosity (Bedford et al , 1993, Proceedings of the 1st Symposium on Enzymes in Animal Nut ⁇ tion, pp 73-77), whereby improved utilization of the plant nutnents by the animal is achieved Thereby, the growth rate and/or feed conversion ratio (/ e , the weight of ingested feed relative to weight gam) of the animal is improved
• the polypeptides of the present invention may also be used in the paper and pulp industry, inter alia in bleaching processes to enhance the brightness of bleached pulps whereby the amount of chlorine used in the bleaching stages is reduced, and to increase the freeness of pulps in the recycled paper process (Enksson, 1990, Wood Science and Technology 24 79-101, Paice et al , 1988, Biotechnol and Bioeng 32 235-239, and Pommier et al , 1989, Tappi Journal 187-191)
• the polypeptides may be used for treatment of lignocellulosic pulp so as to improve the bleachability thereof
• the treatment of lignocellulosic pulp may be performed, for example, as descnbed in U S Patent No 5,658,765, WO 93/08275, WO 91/02839, and WO 92/03608
• the polypeptides of the present invention may also be used in beer brewing, in particular to improve the filterability of wort containing, for example
• the polypeptides of the present invention may be used for separation of components of plant cell materials, in particular of cereal components such as wheat components Of particular interest is the separation of wheat into gluten and starch, i e , components of considerable commercial interest
• the separation process may be performed by use of methods known in the art, conveniently a so-called batter process (or wet milling process) performed as a hydroclone or a decanter process
• the starting material is a dilute pumpable dispersion of the plant material such as wheat to be subjected to separation
• the dispersion is made normally from wheat flour and water
• polypeptides of the invention may also be used in the preparation of fruit or vegetable juice in order to increase yield
• polypeptides of the present invention may also be used as a component of an enzymatic scounng system for textiles
• polypeptides of the present invention may also be used in laundry detergent applications in combination with other enzyme functionalities
• the present invention also relates to nucleic acid constructs comprising a gene encoding a protein, wherein the gene is operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO 2, wherein the gene is foreign to the nucleotide sequence
• the nucleotide sequence comprises or consists of nucleotides 1 to 57 of SEQ ID NO 1
• the present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs
• the present invention also relates to methods of producing a protein comprising (a) cultivating such a recombinant host cell under conditions suitable for production of the protein, and (b) recovering the protein
• the protein may be native or heterologous to a host cell
• the term "protein” is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins
• the term protein * also encompasses two or more polypeptides combined to form the encoded product
• the proteins also include hybrid polypeptides that comprise a combination of partial or complete polypeptide sequences obtained from at least two different proteins wherein one or more (several) may be heterologous or native to the host cell
• Proteins further include naturally occurring allelic and engineered va ⁇ ations of the above mentioned proteins and hybrid proteins
• the protein is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter
• the protein is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase
• the protein is an aminopeptidase, amylase, carbohydrase, carboxypeptida
• Humicola m ⁇ olens DSM 1800 was used as the source of a Family CE1 gene encoding a polypeptide having acetylxylan esterase activity Aspergillus niger M Bin 120 strain (WO 2004/090155) was used for expression of the Humicola insolens gene encoding the polypeptide having acetylxylan esterase activity
• PDA plates were composed per liter of 39 g of potato dextrose agar
• YP medium was composed per liter of 10 g of yeast extract and 20 g of Bacto peptone
• COVE A urea- acetam ⁇ de+ plates were composed per liter of 20 ml of COVE A salts solution, 220 g of sorbitol, 10 g of glucose, 10 ml of 1 M acetamide, and 30 g of Bacto agar, pH 52
• COVE A salts solution was composed per liter of 26 g of KCI, 26 g of MgSO 4 , 76 g of KH 2 PO 4 , and 50 ml of COVE A trace elements solution
• COVE trace elements solution was composed per liter of 0 04 g of Na 2 B 4 O 7 10H 2 O, 04 g of CuSO 4 5H 2 O, 1 2 g of FeSO 4 7H 2 O, 0 7 g of MnSO 4 H 2 O, 08 g of Na 2 MoO 2 2H 2 O, and 10 g of ZnSO 4 7H 2 O
• M410 medium was composed per liter of 50 g of maltose, 50 g of glucose, 2 g of MgSO 4 7H 2 O, 2 g of KH 2 PO 4 4 g of citric acid anhydrous powder, 8 g of yeast extract, 2 g of urea, O 5 g of AMG trace metals solution, and O 5 g of CaCI 2 , pH 6 O
• AMG trace metals solution was composed per liter of 143 g of ZnSO 4 7H 2 O, 25 g of CuSO 4 5H 2 O, O 5 g of NiCI 2 6H 2 O, 138 g of FeSO 4 7H 2 O, 85 g of MnSO 4 7H 2 O, and 3 g of citric acid LB medium was composed per liter of 10 g of tryptone, 5 g of yeast extract, and
• Il Liquid Handling Robot PerkinElmer Life and Analytical Sciences, Boston, MA, USA was used to perform the in-gel digestions A 30 kDa protein gel band was reduced with 50 ⁇ l of 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate pH 8 0 for 30 minutes Following reduction, the gel piece was alkylated with 50 ⁇ l of 55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 80 for 20 minutes The dried gel piece was allowed to swell in 25 ⁇ l of a trypsin digestion solution containing 6 ng of sequencing grade trypsin (Promega, Madison, Wl, USA) per ⁇ l of 50 mM ammonium bicarbonate pH 8 for 30 minutes at room temperature, followed by an 8 hour digestion at 40°C Each of the reaction steps described above was followed by numerous washes and pre-washes with the appropriate solutions following the manufacturer's standard protocol Fifty ⁇ l of acetonit
• a peptide sequence was obtained from a multiple charged peptide ion recovered from the in-gel digested 30 kDa polypeptide gel band
• a doubly charged tryptic peptide ion of 514772 m/z sequence was determined to be Asn-Ser-Tyr-Pro-Gly-Tyr-[Asp or Asn]-Gly-Arg (SEQ ID NO 4)
• SEQ ID NO 4 Example 2: Humicola msolens DSM 1800 genomic DNA extraction
• Humicola msolens DSM 1800 was grown on PDA plates at 45 0 C to confluence Three 4 mm 2 squares were cut from the PDA plates, inoculated into 25 ml of YP medium containing 2% glucose in a baffled 125 ml shake flask, and incubated at 41 0 C with shaking at 200 rpm for 2 days Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, La JoIIa, CA, USA), washed twice in deiomzed water, and frozen under liquid nitrogen Frozen mycelia were ground, by mortar and pestle, to a fine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN I nc , Valencia, CA, USA)
• Example 3 Isolation of a partial fragment of an acetylxylan esterase gene from Humicola insolens DSM 1800
• the amplification reaction (25 ⁇ l) was composed of 117 ng of Humicola msolens DSM 1800 genomic DNA as template, 04 mM each of dATP, dTTP, dGTP, and dCTP, 50 pmol each of primer HiFAE-degR and primer HiFAE-degF, 1X ADVANTAGE® GC- MeIt LA Buffer (Clontech Laboratories, lnc , Mountain View, CA, USA), and 1 25 units of ADVANTAGE® GC Genomic Polymerase Mix
• the amplification was performed using an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, lnc , Westbury, NY,
• reaction products were isolated by 1 0% agarose gel electrophoresis in TBE (108 g of Tns base, 5 5 g of boric acid and 4 ml of 05 M EDTA pH 80 per liter) buffer
• a PCR product band of approximately 1 1 kb was excised from the gel, purified using a QIAQUICK® Gel Extraction Kit (QIAGEN lnc , Valencia, CA, USA) according to the manufacturer's instructions, and sequenced Based on the sequencing it was found that primer HiFAE-degF did not bind during the amplification, while primer HiFAE-degR bound to both ends
• a partial sequence was obtained which encoded a peptide fragment that was homologous to a putative acetylxylan esterase from Neosartorya nsche ⁇ (Uniprot A1 DBP9)
• Example 4 Identification of a full-length Humicola insolens acetylxylan esterase gene
• a full-length Family CE1 acetylxylan esterase gene was identified from Humicola insolens DSM 1800 using a GENOMEWALKERTM Universal Kit (Clontech Laboratories, lnc , Mountain View, CA, USA) according to the manufacturer's instructions Briefly, total genomic DNA from Humicola insolens DSM 1800 was digested separately with four different restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) that leave blunt ends Each batch of digested genomic DNA was then ligated separately to the GENOMEWALKERTM Adaptor (Clontech Laboratories, lnc , Mountain View, CA, USA) to create four libraries These four libraries were then employed as templates in PCR reactions using two gene-specific p ⁇ mers shown below, one for a primary PCR and one for a secondary PCR amplifying downstream of the fragment through the 3' end encoding the C-term ⁇ us of the acetylxylan esterase Based on sequence homology to
• Primer H ⁇ ns_AXE_GSP2_F3 (secondary) 5 1 -ACACTGGGCCAGGACGGCGCTCGATAT-3 1 (SEQ ID NO 9)
• the primary amplifications were composed of 1 ⁇ l (approximately 6 ng) of each library as template, 04 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmol of Adaptor Primer 1 (Clontech Laboratories, lnc , Mountain View, CA, USA), 50 pmol of primer H ⁇ ns_AXE_GSP1_F1, 1X ADVANTAGE® GC-MeIt LA Buffer (Clontech Laboratories, lnc , Mountain View, CA, USA), and 1 25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of 25 ⁇ l The amplifications were performed
• the secondary amplifications were composed of 1 ⁇ l of each primary PCR product as template, 04 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmol of Adaptor Primer 2 (Clontech Laboratories, lnc , Mountain View, CA, USA), 50 pmol of primer H ⁇ ns_AXE_GSP2_F3, 1X ADVANTAGE® GC-MeIt LA Buffer, and 1 25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of 25 ⁇ l
• the amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denatu ⁇ ng at 95°C for 1 minute, 5 cycles each at a denaturing temperature of 95°C for 25 seconds, annealing and elongation at 72°C for 5 minutes, 20 cycles each at a denatunng temperature of 95 0 C for 25 seconds, annealing and elongation
• reaction products were isolated by 1 0% agarose gel electrophorsis in TBE buffer From the Pvu Il library, 1 kb and 1 8 kb products were excised from the gel, purified using a QIAQUICK® Gel Extraction Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions, and sequenced
• DNA sequencing of the PCR fragments was performed with a Perkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer using dye-terminator chemistry (Giesecke ef a/ , 1992, supra) and primer walking strategy Adaptor Primer 2 and primer H ⁇ ns_AXE_GSP2_F3 were used for sequencing Nucleotide sequence data were scrutinized for quality and all sequences were compared to each other with assistance of PHRED/PHRAP software (University of Washington, Seattle, WA, USA) The PCR fragment sequence results were compared and aligned with the partial acetylxylan esterase gene sequence from Humicola insolens descnbed in Example 3 A gene model was constructed based on the gene fragments obtained in this Example and in Example 3 allowing determination of the 5' and 3' ends of the gene with other homologous acetylxylan esterases
• Example 5 Cloning of the Humicola insolens acetylxylan esterase gene and construction of an Aspergillus niger expression vector
• Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Humicola insolens acetylxylan esterase gene from the genomic DNA prepared in Example 2
• An InFusion Cloning Kit (BD Biosciences, Palo Alto, CA, USA) was used to clone the fragment directly into the expression vector pBM120a (WO 2006/078256) HinsAXEBDinfnterm ⁇ '-ACACAACTGGCCATGAAGGTCCCGACTCTCATCTCG-S' (SEQ ID NO 10) HinsAXEBDinfCtermendPacl
• reaction products were isolated by 1 0% agarose gel electrophoresis in TBE buffer where an approximately 1 2-1 3 kb product band was excised from the gel, and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions
• Plasmid pBM120a was digested with Nco I and Pac I, isolated by 1 0% agarose gel electrophoresis in TBE buffer, and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions
• the gene fragment and the digested vector were ligated together using an InFusion Cloning Kit resulting in pMMar ⁇ ( Figure 2) in which transcription of the acetylxylan esterase gene was under the control of a hybrid of promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase (NA2-tp ⁇ promoter)
• the ligation reaction (20 ⁇ l) was composed of 1X InFusion Buffer (BD Biosciences, Palo Alto, CA, USA), 1X BSA (BD Biosciences, Palo Alto, CA, USA), 1 ⁇ l of InFusion enzyme (diluted 1 10) (BD Biosciences, Palo Alto, CA, USA), 106 ng of pBMi20a digested with Nco I and Pac I, and 208 ng of the purified Humicola insolens acetylxylan esterase PCR product The reaction was incubated at room temperature
• Plasmid pMMar ⁇ was isolated from broth using a QIAGEN® Midi Kit according to the manufacturer's instructions Plasmid pMMar ⁇ was digested with Pme I, isolated by 1 0% agarose gel electrophoresis in TBE buffer, and the fragment containing the acetylxylan esterase gene was purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions in preparation for transforming Aspergillus niger MB ⁇ n120 protoplasts The same approximately 1 2-1 3 kb PCR fragment was cloned into pCR®2 1-TOPO® vector (Invitrogen, Carlsbad, CA, USA) using a TOPO® TA CLONING Kit (Invitrogen, Carl
• Example 6 Characterization of the Humicola insolens genomic sequence encoding a Family CE1 acetylxylan esterase
• Nucleotide sequence data (Example 5) were scrutinized for quality and all sequences were compared to each other with assistance of PHRED/PHRAP software (University of Washington, Seattle, WA, USA)
• the nucleotide sequence (SEQ ID NO 1) and deduced ammo acid sequence (SEQ ID NO 2) are shown in Figures 1A and 1B
• the genomic fragment encodes a polypeptide of 377 amino acids, interrupted by 2 predicted introns of 73 bp and 62 bp
• the % G+C content of the full-length coding sequence and the mature coding sequence are 604% and 605%, respectively
• SignalP software program (Nielsen et al , 1997, Protein Engineering 10 1-6)
• the predicted mature protein contains 358 amino acids with a molecular mass of 385 kDa
• a predicted esterase polyhydroxybutyrate depolymerase domain occurs at amino acids 43 to 257 and a predicted cellulose-binding domain at amino acids 341 to 377
• the acetylxylan esterase appears to fall into the carbohydrate esterase Family CE1 according to Coutinho and Hennssat, 1999,
• Example 7 Transformation and expression of the Humicola insolens Family CE1 acetylxylan esterase gene in Aspergillus niger MBini 20
• the Humicola insolens Family CE1 acetylxylan esterase gene was expressed in Aspergillus niger MB ⁇ n120 Aspergillus niger MB ⁇ n120 protoplasts were prepared according to the method of Christensen ef a/ , 1988, Bio/Technology 6 1419-1422 Five ⁇ g of Pme I digested pMMar ⁇ were used to transform Aspergillus niger MBiM 20 The transformation of Aspergillus niger UBm 120 with the Pme I digested pMMar ⁇ yielded approximately 45 transformants Twenty-five transformants were isolated to individual COVE A urea- acetam ⁇ de+ plates Two 3 mm square agar plugs were cut from the confluent COVE A urea- acetam ⁇ de+ plates of the 25 transformants and inoculated separately into 25 ml of M410 medium in 125 ml plastic shake flasks and incubated at 34°C with shaking at 250
• Shake flask medium was composed per liter of 70 g of sucrose and 100 g of soy concentrate Trace metals solution was composed per liter of 13 8 g of FeSO 4 7H 2 O, 143 g of ZnSO 4 7H 2 0, 11 6 g of MnSO 4 H 2 O, 25 g of CuSO 4 5H 2 O, 05 g of NiCI 2 6H 2 O and 33 g of crt ⁇ c acid monohydrate
• One hundred ml of shake flask medium were added to each of four 500 ml shake flasks The shake flasks were each inoculated with 200 ⁇ l from a glycerol spore stock of Aspergillus niger MMar204 and incubated at 30°C on an orbital shaker at 220 rpm for 72 hours
• Fifty ml of the shake flask broth from each of the four shake flasks were used to inoculate a 3 liter fermentation vessel
• Fermentation batch medium was composed per liter
• a total of 2 liters of the fermentation batch medium was added to an Applikon Biotechnology two liter glass jacketed fermentor (Applikon Biotechnology, Schiedam, Netherlands) Fermentation feed medium was dosed at a rate of O to 4 g/l/hr for a period of 185 hours
• the fermentation vessel was maintained at a temperature of 34 0 C and pH was controlled using an Applikon 1030 control system (Applikon Biotechnology, Schiedam, Netherlands) to a set-point of 5 1 +/- O 1 Air was added to the vessel at a rate of 1 wm and the broth was agitated by Rushton impeller rotating at 1100 rpm
• whole broth was harvested from the vessel and centrifuged at 3000 x g to remove the biomass
• the supernatant was sterile filtered and stored at 5 to 1O 0 C
• Example 9 Purification of the Humicola insolens polypeptide having acetylxylan esterase activity
• Supernatant from the fermentation broth desc ⁇ bed in Example 8 was first buffer- exchanged into 20 mM MES pH 6 and concentrated using a Pall Filtron tangential flow filtration system consisting of an Ultrapump II, an ULTRARESERVOIRTM 5L, and an ULTRASETTETM 10K Omega tangential flow filtration membrane with a 10,000 Da molecular weight cut-off (Pall Corporation, East Mills, NY, USA)
• the resulting buffer- exchanged material 150 ml
• SP SEPHAROSETM Big Beads resin GE Healthcare, Piscataway, NJ, USA
• Fractions were collected and monitored at 280 nm A 25 ⁇ l aliquot of the fractions having UV absorbance at 280 n
• the Humicola insolens acetylxylan esterase was determined to have an activity of 154 units per mg of enzyme
• Example 10 Thermostability of Humicola insolens acetylxylan esterase
• the thermostability of the purified Humicola insolens acetylxylan esterase was determined by differential scanning calorimetry (DSC) using a A VP- DSC (MicroCal lnc , Northampton, MA, USA) according to the manufacturer's instructions in 50 mM sodium acetate pH 50
• the thermal denaturation temperature, Td was taken as the top of the denaturation peak (major endothermic peak) in a thermogram (Cp vs T) obtained after heating of the enzyme solution at a programmed heating rate of 90 0 C per hour beginning at 2O 0 C
• the Td for the acetylxylan esterase under these conditions was
• Example 11 Effect of Humicola insolens acetylxylan esterase on hydrolysis of p retreated com fiber
• Corn fiber is a fraction from the wet milling of corn kernels Com fiber is the seed coat and residual endosperm left after starch is removed and further processed Corn fiber was pretreated by autoclaving at 140 0 C for 150 minutes The amount of arabinose, glucose and xylose in the substrate was determined to be 175, 317, and 261 g per kg dry matter using the following methods
• Arabinose and xylose were determined by carbohydrate hydrolysis using dilute hydrochloric acid
• the pretreated corn fiber was transferred to 125 ml conical flasks and diluted to contain approximately 10% dry matter
• the corn fiber sample was preheated at 100 0 C in an oil bath Hydrolysis was started by adding 5 ml of 2 M hydrochloric acid for 2 hours at 100 0 C After incubation the flasks were cooled on ice and neutralized with 4 M sodium hydroxide Samples were filtered with a M I N I S ART® 02 micron syringe filter (Sartorius AG, Goettingen, Germany) and analyzed for arabinose and xylose on a DlONEX BIOLC® System (Dionex Corporation, Sunnyvale, CA, USA) Glucose was determined by subjecting the pretreated sample of corn fiber to a two step sulfunc acid hydrolysis Three ml of 72% sulfuric acid was added to approximately 300 mg of dned corn fiber
• the hydrolysis of the pretreated corn fiber was conducted with a Tnchoderma reesei cellulolytic protein composition (Tnchoderma reesei broth comprising Thermoascus aurantiacus GH61A polypeptide having cellulolytic enhancing activity and Aspergillus oryzae beta-glucosidase fusion, PCT/US2008/065417) and a Tnchoderma reesei beta-xylosidase
• the Tnchoderma reesei beta-xylosidase was obtained recombinant ⁇ by expression in Aspergillus oryzae as described in Rasmussen et al , 2006, Biotechnology and Bioengineenng 94 869-876 using standard cultivation methods for Aspergillus oryzae
• the Humicola insolens acetylxylan esterase was obtained as described in Example 9
• the hydrolysis of the pretreated com fiber was performed in 2 ml
• Example 12 Effect of Humicola insolens acetylxylan esterase on the hydrolysis of D-xylose tetraacetate
• D-xylose tetraacetate (Benn Chemicals, Dielsdorf, Switzerland) was performed in 1 5 ml EPPENDORF® tubes at a temperature of 5O 0 C and a pH of 5 0 in 50 mM succinic acid for 48 hours Samples were incubated in a THERMOMIXER® Comfort that subjected each sample with constant heating and mixing The substrate amount used was 1 ml at a concentration of 1 w/w % of D-xylose tetraacetate The control sample (1 ml of substrate) was compared with the Humicola insolens acetylxylan esterase sample (1 ml of substrate + 7 ⁇ l of enzyme) The Humicola insolens acetylxylan esterase was added at an enzyme loading of 05 mg Humicola insolens acetylxylan esterase/g dry solids Hydrolysis was terminated after 48 hours by heating the samples for 10 minutes at 100 0 C in a heat block
• the strain has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto
• the deposit represents a substantially pure culture of the deposited strain
• the deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action
• polypeptide of paragraph 2 comprising an amino acid sequence having at least 80% identity to the mature polypeptide of SEQ ID NO 2
• polypeptide of paragraph 3 comprising an amino acid sequence having at least 85% identity to the mature polypeptide of SEQ ID NO 2
• polypeptide of paragraph 4 comprising an amino acid sequence having at least 90% identity to the mature polypeptide of SEQ ID NO 2
• polypeptide of paragraph 5 comprising an amino acid sequence having at least 95% identity to the mature polypeptide of SEQ ID NO 2
• the polypeptide of paragraph 8 comprising or consisting of the mature polypeptide of SEQ ID NO 2 [11]
• the polypeptide of paragraph 1 which is encoded by a polynucleotide that hyb ⁇ dizes under at least medium-high stringency conditions with ( ⁇ ) the mature polypeptide coding sequence of SEQ ID NO 1 , ( ⁇ i) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1, or (in) a full-length complementary strand of ( ⁇ ) or ( ⁇ i) [12]
• the polypeptide of paragraph 11 which is encoded by a polynucleotide that hyb ⁇ dizes under at least high stringency conditions with ( ⁇ ) the mature polypeptide coding sequence of SEQ ID NO 1, (n) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1 , or (in) a full-length complementary strand of ( ⁇ ) or (n) [13]
• polypeptide of paragraph 13 which is encoded by a polynucleotide comprising a nucleotide sequence having at least 80% identity to the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 14 which is encoded by a polynucleotide comprising a nucleotide sequence having at least 85% identity to the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 15 which is encoded by a polynucleotide comprising a nucleotide sequence having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 16 which is encoded by a polynucleotide comprising a nucleotide sequence having at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 17 which is encoded by a polynucleotide comprising a nucleotide sequence having at least 97% identity to the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 1 which is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO 1, or a subsequence thereof encoding a fragment having acetylxylan esterase activity
• polypeptide of paragraph 19 which is encoded by a polynucleotide comprising or consisting of the mature polypeptide coding sequence of SEQ ID NO 1
• polypeptide of paragraph 1 wherein the polypeptide is a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO 2
• polypeptide of any of paragraphs 1-24, wherein the mature polypeptide coding sequence is nucleotides 58 to 1266 of SEQ ID NO 1
• a nucleic acid construct comprising the polynucleotide of paragraph 26 or 27 operably linked to one or more (several) control sequences that direct the production of the polypeptide in an expression host
• a recombinant host cell comprising the nucleic acid construct of paragraph 28
• a method of producing the polypeptide of any of paragraphs 1-25 comprising (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide, and (b) recovenng the polypeptide
• a method of producing the polypeptide of any of paragraphs 1-25 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide, and (b) recovering the polypeptide
• a method of producing a mutant of a parent cell comprising disrupting or deleting a nucleotide sequence encoding the polypeptide of any of paragraphs 1-25, which results in the mutant producing less of the polypeptide than the parent cell
• mutant cell of paragraph 34 further comprising a gene encoding a native or heterologous protein
• a method of producing a protein comprising (a) cultivating the mutant cell of paragraph 35 under conditions conducive for production of the protein, and (b) recovenng the protein
• the isolated polynucleotide of paragraph 26 or 27, obtained by (a) hybridizing a population of DNA under at least medium-high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO 1 , ( ⁇ i) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1 , or ( ⁇ ) a full- length complementary strand of ( ⁇ ) or ( ⁇ i), and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having acetylxylan esterase activity
• the isolated polynucleotide of paragraph 37 obtained by (a) hybridizing a population of DNA under at least high stringency conditions with ( ⁇ ) the mature polypeptide coding sequence of SEQ ID NO 1 , ( ⁇ ) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO 1, or (in) a full-length complementary strand of ( ⁇ ) or ( ⁇ ), and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having acetylxylan esterase activity [39] The isolated polynucleotide of paragraph 37 or 38, wherein the mature polypeptide coding sequence is nucleotides 58 to 1266 of SEQ ID NO 1
• a method of producing a polynucleotide comprising a mutant nucleotide sequence encoding a polypeptide having acetylxylan esterase activity comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO 1 , wherein the mutant nucleotide sequence encodes a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO 2, and (b) recovering the polynucleotide comprising the mutant nucleotide sequence
• a method of producing a polypeptide comprising (a) cultivating a cell comprising the mutant polynucleotide of paragraph 41 encoding the polypeptide under conditions conducive for production of the polypeptide, and (b) recovering the polypeptide
• a method of producing the polypeptide of any of paragraphs 1-25 comprising (a) cultivating a transgenic plant or a plant cell comp ⁇ sing a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide, and (b) recovering the polypeptide
• a double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of paragraph 26 or 27, wherein optionally the dsRNA is a siRNA or a miRNA molecule
• dsRNA double-stranded inhibitory RNA
• paragraph 45 which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length
• dsRNA double-stranded inhibitory RNA
• a recombinant expression vector comprising the nucleic acid construct of paragraph 49
• a recombinant host cell comprising the nucleic acid construct of paragraph 49
• a method of producing a protein comprising (a) cultivating the recombinant host cell of paragraph 51 under conditions conducive for production of the protein, and (b) recovering the protein
• a method for degrading a xylan-containing material comprising treating the xylan-containi ⁇ g material with the polypeptide having acetylxylan esterase activity of any of paragraphs 1-25 [54] The method of paragraph 53, further comprising treating the xylan- containi ⁇ g material with a xylan degrading enzyme

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• 2008
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